The primary goal of this Biomedical Research Grant is to translate imaging agents for intraoperative cancer detection and image-guided surgery. Half of all cancer patients undergo surgery to remove their tumor. The single most important predictor of patient survival is a complete surgical resection of the primary tumor, draining lymph nodes, and metastatic lesions. However, up to 40% of surgical patients leave the operating room with malignant cells remaining after resection. Thus, there are urgent unmet needs to develop new and innovative technologies that can assist the surgeon in ensuring complete tumor resection by delineating tumor margins, and identifying micrometastases and draining lymph nodes. We propose a new technology to image patients at the conclusion of the standard-of-care cancer operation in order to discover residual local disease before completing the surgery. While the proposed technologies are broadly applicable to solid tumors, this project is specifically committed to improving detection of lung adenocarcinomas, one of the most aggressive human malignancies. To accomplish this goal, we have assembled a collaborative team at University of Pennsylvania and Purdue University with synergistic expertise in clinical trials, folate biology, chemistry, lung pathology, thoracic surgery and veterinary medicine. The proposed work will optimize three distinct types of contrast agents for a translational intraoperative imaging clinical trial. The first contrast agent uses folate to target a conjugated NIR dye specifically to malignant tissue. The folate-NIR agent will be evaluated in a first-in- human clinical trial of lung adenocarcinoma patients in order to determine if there is a benefit of intraoperative imaging to standard-of-care surgical approaches. The second contrast agent uses the same targeting approach to deliver a near-infrared dye for EGFR and CCK2R to cancer cells and allow better imaging of diseased cells more deeply buried in the tissues. This will allow for precise tumor localization and more accurate resection of cancers without violating the tumor capsule or seeding the body cavity.